1. Field of the Invention
This invention relates to the field of flow cytometry and, more particularly, to improvements in apparatus and methods for detecting back-scatter (i.e., radiation scattered or reflected backwardly) from irradiated particles passing through an optical flow cell of a flow cytometer.
2. The Prior Art
The use of light-scattering (LS) measurements as a means for differentiating various types of small particles, e.g., blood cells, in a liquid suspension is well known. For example, in virtually all sophisticated flow hematology instruments and fluorescence flow cytometers, it is common to measure the forward light scattering properties of individual blood cells by passing the cells, one at a time, through the “interrogation zone,” i.e., the constricted region, of an optically transparent flow cell. While positioned within such interrogation zone, each blood cell is irradiated by a focused laser beam, and one or more photodetectors, strategically positioned forward of the interrogation zone, operate to sense the level of forward-scattered radiation, usually within different predetermined angular ranges. In addition to detecting a portion of the forward-scatter signature of the irradiated cells, some cytometric instruments measure side-scatter as well, using a separate photodetector located laterally of the irradiated cells to radiation scattered at approximately 90 degrees to the irradiating beam. These light-scattering measurements are often combined with other simultaneously made measurements, e.g., axial light-loss, DC volume and/or RF conductivity measurements, to better differentiate particles and cells of particular interest from other cells and particulate material in the sample having similar forward and side-scattering properties within the measurement ranges. See, for example, the disclosure of the commonly assigned U.S. Pat. Nos. 5,125,737, and 6,228,652, both being issued in the names of Rodriguez et al. Having made various different parametric measurements on each cell, the cytometric instrument produces scattergrams in which the different parameters measured are plotted against each other. Ideally, each cell or particle type appears on these scattergrams as a tight cluster of data points, each point representing an individual cell or particle in the sample, and each cluster being readily identifiable from other clusters by a clearly identified spacing that separates various clusters in the scattergram. In such case, it is a relatively simple matter to “gate” cells of one cluster from those of another cluster and to enumerate the cells of each cluster. This ideal, unfortunately, is sometimes difficult to realize since, for a variety of reasons, a certain percentage of cells of one type invariably invade the spatial domain of cells of other types, making the differentiation of each type somewhat imprecise.
To more precisely differentiate certain blood cells and the like on the basis of their light-scattering signature, it has been suggested to further determine a portion of the back-scatter signature (i.e., the intensity-distribution of radiation (light) reflected back towards the irradiating source) of such cells. In the commonly assigned U.S. application Ser. No. 10/227,004 filed in the name of D. Kramer on Aug. 23, 2002, a back-scatter collector/detector is disclosed that is adapted for use in a conventional flow cytometer to collect and detect a portion of the back-scattered light from an irradiated cell as it passes through an optical flow cell. The light-collecting component of such a device comprises a plurality of optical fibers, each having a light-collecting end supported by a housing that serves to arrange such ends in a circular array. A central bore hole in the fiber-supporting housing enables the cell-irradiating laser beam to pass uninterrupted through the housing (and the center of the fiber array) as the beam propagates from its source towards the cell interrogation zone of the flow cell. The light-discharge end (opposite the light-collecting end) of each optical fiber is optically coupled to a high-gain photo-detector, e.g., a photomultiplier tube, which provides a signal indicative of the level of light collected by the fiber ends. It may be appreciated that the angle at which the back-scatter light is detected from each cell is determined by both the diameter of the circular array of fiber ends, and the displacement between the scatter source, which is nominally the center of the cell interrogation zone, and the fiber ends. As the diameter of the array increases and/or the displacement between the flow cell center and the fiber ends decreases, the back-scatter angle of detection increases, and vice versa.
In light-scatter measurement systems of the above type, the cell-irradiating laser beam is typically brought to focus within the cell-interrogation zone by a beam-shaping lens system located on the beam axis between the light beam source and the flow cell. Such a lens system usually comprises a pair of spaced plano-convex lenses having perpendicularly-crossed optical powers. By this arrangement, one lens serves to focus the laser beam in a first plane while the other serves to focus the laser in a second plane perpendicular to the first plane, whereby a desired elliptical focus pattern is achieved. The two lenses are commonly supported in a desired spaced relationship, typically about 50 to 70 mm. apart, within the barrel of a cylindrical housing. The size of the focus spot is determined by the respective focal lengths of the lenses. Different spot sizes afford different advantages. For example, larger spots may be useful in differentiating relatively large cells, and smaller spots may be required to achieve a relatively high flux density, as may be necessary to excite certain fluorochromes to which certain cells of interest have been previously coupled for detection. To achieve a small spot size, the focal length of the front lens (i.e., the lens closer to the flow cell) must be relatively short which, unfortunately, reduces the space between the front lens and the front wall of the flow cell wall. Typically, the front lens of the optical system has a focal length of about 10 to 15 mm., and the rear lens (i.e., the lens further from the flow cell) has a focal length of about 60 to 80 mm.
In the above-noted Kramer application, the optical fibers of the back-scatter collector are arranged in a relatively tiny circular array having a diameter of only about 1.75 mm. Each optical fiber in the array has a nominal diameter of 500 microns, and there are about 20 fibers in the array. As described, the back-scatter collector is to be positioned within the above-noted space between the front lens of the laser-focusing system and the front wall of the optical flow cell. While the disclosed back-scatter collector can be readily positioned within this space when the focal length of the front lens is 15 mm. or greater, proper placement of the collector becomes considerably more difficult (if not impossible) as the focal length of the front lens of the focus system approaches 10 mm. It will be appreciated that the distance between the center of the flow cell's interrogation zone and the outside wall of the flow cell consumes a portion of the focal length distance. Thus, though the focal length of the rear lens may be 10 mm., the actual space in which the back-scatter collector may be positioned will be somewhat shorter. Further, even were it possible to position the circular array of fiber ends of a back-scatter collector of the type described above within an axial space as short as 10 mm., the angle at which back-scatter would be detected by the circular array of fibers can become excessively large (due the close spacing between the array and the center of the cell-interrogation zone of the flow cell).